Characterisation of three mosquitocidal Bacillus strains isolated from mangrove forest

In an attempt to isolate mosquitocidal bacteria, 460 samples of soil, leaf and water were collected from mangrove habitats of Andaman–Nicobar Islands, India. Out of a total number of 857 bacteria screened, three Bacillus strains showed mosquitocidal activity at promising levels. Two of them were identified, morphologically and biochemically, as Bacillus subtilis, whereas one was Bacillus thuringiensis. These strains were code named as B469, B471 and B474, respectively. Molecular characterisation using 16S rRNA confirmed the identity of B469 and B471 as B. subtilis strains and flagellar serotyping confirmed B474 as B. thuringiensis subsp. israelensis/tochigiensis (H14/H19). Amplification of cry and cyt genes of B474 showed the presence of cry 4A, cry 4B, cry 11, cry 10, cyt 1 and cyt 2. Bioassay of the culture supernatants (CS) of B. subtilis strains showed mosquitocidal activity against larvae and pupae of Anopheles stephensi, Culex quinquefasciatus and Aedes aegypti. The mosquitocidal activity was more pronounced with the CS of B471 than that of the B469. Interestingly, CS of both these strains was more active on pupae, a non-feeding stage, than larvae of the mosquitoes tested. The B. thuringiensis strain was able to kill the larvae of all the above mentioned mosquitoes. This is the first study demonstrating mosquito pupicidal activity of B. subtilis strain. Moreover, this is the first report of isolation of a highly active B. thuringiensis subsp israelensis/tochigiensis from India.